We obtained the following results during the term of this project. 1.Gene Introduction into Animal Tissues Using Multilamella Fusogenic Liposomes.We prepared multilamella fusogenic liposomes encapsulated DNA and protein by incubating liposomes with Sendai virus and purifying them on sucrose gradient. These liposomes could introduce and express E.coli beta-galactosidase gene and the surface antigen gene of human hepatitis B virus into liver cells of living rats. 2.Purification and Characterization of Singlelamella Fusogenic Liposomes.We prepared singlelamella fusogenic liposomes encapsulating DNA and protein by preparing liposomes by reverse phase method, incubating them with Sendai virus and purifying them on sucrose gradient. We found that these liposomes were essentially free from unfusogenic liposomes and virus and that they had higher efficiency of introduction of macromolecules into cells(10^<10> particles for protein and 10^7 particles for DNA per 1 OD_<540> unit). The efficiency
… More of these purified liposomes to introduce their contents into cytoplasm was the same as that of intact Sendai virus. We also found that these liposomes had a diameter of 250nm. 3.Analysis of Mechanism of Persistent Infection of Mutant Sendai Virus.We demonstrated the ts mutant, Cl151, Caused persistent infection because their mutant M protein was unstable at non-permissive temperature. We also showed that M protein did not affect the gene expression of the virus. This is a first step for the development of the virus vector based on Sendai virus. 4.Development of the Vectors for Human Artificial Chromosome.We succeeded to make the vectors construct the human artificial chromosome. The vectors(pMYAC1,pMYAC2) consisted of human telomere, yeast DNA for maintaining as yeast artificial chromosome, drug resistant genes as selection markers in human cells. This is the first step for construction of human artificial chromosome, an ultimate system for human gene therapy.3.ヒト人工染色体の構築に向けた基礎研究。DNA合成をしていない細胞で遺伝子を安定化させるために細胞内で染色体して存在できるDNA構造の研究を行った。本研究では合成したヒト・テロメアを使ってこのために必要な人工染色体クローニング・ベクターの構築に成功した。 Less